Project Summary/Abstract The astrocytic tumor glioblastoma multiforme (GBM) is an incurable and highly aggressive malignancy of the brain that was recently found to form an interconnected, gap junction-mediated network. These gap junctions propagate intercellular calcium waves (ICWs) across tumor cells and promote tumor growth, but the biological mechanisms underlying this growth remain unknown. Gap junctions transmit many small molecules, such as calcium, that have the potential to alter metabolic and signaling pathways, which may promote tumor growth. Understanding the regulation and consequences of this intercellular signaling may therefore provide insight into how this network promotes tumor growth. Similar to GBM tumors, normal astrocytes also transmit ICWs across gap junctions, suggesting that related mechanisms may regulate ICWs in astrocytes and GBM. Glutamate can stimulate ICWs in astrocytes and is a known promoter of GBM growth, so studies outlined in Aim 1 will characterize whether glutamate also induces ICWs in GBM and whether this induction is an important aspect of glutamate-mediated GBM growth. This work will utilize a novel model of GBM wherein human glioblastoma stem cells (GSCs) form GBM tumors in human cerebral organoids. The GSCs will express genetically encoded calcium indicators in order to visualize ICWs basally and in response to glutamate. ICWs will then be inhibited during tumor growth to observe whether glutamate can promote tumor growth independent of ICW propagation. Increased calcium levels can promote the uptake of glucose in astrocytes, so gap-junction mediated transmission of ICWs may similarly promote glucose uptake in GBM, promoting tumor growth. The studies described in Aim 2 will determine whether the presence of gap junctions and ICWs alter glucose metabolism in GBM. GBM tumors with and without Connexin 43 (Cx43), the gap junction protein responsible for ICW propagation, will be grown in human cerebral organoids. Tumor ability to take up glucose and translocate GLUT1 to the cell surface will be analyzed, and metabolic flux analysis will be conducted to determine whether gap junctions alter glucose utilization within the tumor. As small molecules transmitted through gap junctions may also regulate signaling pathways, studies in Aim 3 will explore whether the presence of gap junctions induces signaling changes that promote GBM growth through comparison of the phosphoproteomes of GBM tumors with and without Cx43. In exploring the hypothesis that gap junctions promote GBM growth through the transmission of molecules that regulate metabolic and signaling pathways, this study aims to identify therapeutically relevant targets in the treatment of GBM.